摘要
【目的】克隆巴西橡胶抗病基因同源序列，为巴西橡胶抗病R基因的获取奠定基础。【方法】根据已知抗病基因（Resistance gene， R gene）编码的蛋白质NBS-LRR保守结构设计一对简并引物 P1/P2，采集经水杨酸（SA）处理0、12、24、48 h的巴西橡胶叶片，进行cDNA同源序列克隆。【结果】序列分析结果表明，克隆获得的RGAs经系统进化分析分为TIR-NBS-LRR和non-TIR-NBS-LRR 两种类型和9个基因家族；多重比较发现，克隆获得的RGAs具有典型的NBS类抗病基因保守结构域，在与已知植物NBS类序列相似性比较中，与I2c-2基因相似性最高，为46.2%，而巴西橡胶NBS类RGAs之间氨基酸相似性为22.1%～100.0%。进一步研究发现，核苷酸非同义替换和同义替换的比率小于1，有低水平的重组，表明点突变逐步积累是导致纯化选择的主要力量。【结论】通过SA诱导作用和NBS类抗病基因结合得到的巴西橡胶NBS类RGAs具有广泛的遗传多样性，并与已知抗病基因氨基酸序列具高度相似性，这些RGAs候补序列可能与某抗病基因有密切联系。
【Objective】The present study aimed to clone the nucleotide binding site （NBS） type resistance gene analogs （RGAs） in Hevea brasiliensis in order to provide references for acquisition of resistance genes （R genes）. 【Method】The resistance gene analogs were cloned from the cDNA of leaf by SA induction for respective 0, 12, 24, 48 h using a pair of specific degenerated primers based on the conserved regions of NBS-leucine rich repeat （LRR） domain of plant R genes. 【Result】Sequence analysis showed that the cloned Hevea brasiliensis RGAs belonged to the non-TIR-NBS-LRR and TIR-NBS-LRR type and 9 gene families. Through multiple comparisons, the cloned RGAs had typical conservative domain of NBS. Compared with known NBS sequence, RGAs had similarity rate of 46.2% with I2c-2 gene, while amino acid similarity amongst RGAs in Hevea brasiliensis NBS reached 22.1%-100.0%. Further studies revealed the ds/dn ratio was less than 1 indicating a low level of recombination. NBS analogs in Hevea brasiliensis were subject to gradual accumulation of mutations which was the major force leading to purifying selection. 【Conclusion】Obtained from SA induction and resistance gene NBS, the cloned RGAs had wide heredity diversity and high similarity with amino acid sequence in known resistance gene. Those candidate resistance gene sequences may be closely related to a resistance gene in Hevea brasiliensis.
出处
《南方农业学报》
CAS
CSCD
北大核心
2013年第3期371-376,共6页
Journal of Southern Agriculture
基金
海南大学"211工程"热带作物遗传育种与生态保育创新人才培养基金项目(YJS-2011-0210)
作者简介
游启孙（1987-），研究方向为橡胶树育种种质资源及分子生物学，E-mail：704015097@qq.com
为通讯作者，莫廷辉（1964-），副教授，主要从事橡胶学及作物遗传育种研究工作，E-mail：31112409@163.com。
